Growth of pneumococcal cells is limited under iron-limited conditions and the growth can be restored by addition of either hemin or hemoglobin, but not by transferrin or lactoferrin. Mice injected with a pneumococcal mutant defective in hemin utilization survived longer than mice receiving wild-type cells, suggesting the ability to utilize hemin is related to the pneumococcal virulence. Pneumococcal cells showed a great capability to bind hemin, and the level of binding activity was not affected by iron source supplemented in the growth medium. The hemin binding activity was mediated by protein(s) located in the soluble extract and cell membrane fractions. A major hemin binding polypeptide with an apparent molecular weight of 43 kDa was identified and isolated from the pneumococcal cell lysate. By immunoblot analysis, this hemin binding polypeptide was localized in the membrane. The subcellular distribution of hemin binding activity may have functional implications.